Cytidine VOLUME

In humans, the mean maximum plasma concentration (Cmax) is 79 ng/mL and the time to achieve Cmax was 2.67 h after a single 3-h infusion of decitabine (1) at 15 mg/m2. At this dose, the volume of distribution at steady state is 148 mL/kg, and the total plasma clearance is 122 L/kg/m2. The terminal half-life is approximately 35 min as decitabine (1) is primarily metabolized in the liver by cytidine deaminase to yield noncytotoxic 5-aza-2 -deoxyuridine. Urinary excretion of unchanged decitabine (1) is low (0.01-0.9% of total dose).13... 

The standard assay mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM dithiothreitol, 0.1 to 10 mM substrate, and the appropriate amount of enzyme in a final volume of 500 nL. Cytidine monophosphate (CMP) at an initial concentration of 0.2 mM was used to assay red blood cell lysates. Incubations were carried out at 37°C for either 30 or 60 minutes before the reaction was stopped by adding 100 /u.L of assay mixture to 50 fiL of ice-cold 1.2 M perchloric acid. After the tubes had cooled for 10 minutes in an ice bath, proteins were removed by centrifugation and 130 /u.L of the supernate was neutralized by the addition of 35 /u,L of 1 M K2C03. Potassium perchlorate was removed by centrifugation before analysis by HPLC. 

Figure 9.89 Pyrimidine 5 -nucleotidase assay in undialyzed human erythrocyte lysate obtained by 1 5 dilution of packed cells with deionized water, (a) Separation of 1 nmol of CMP, cytidine, and uridine as the standards. Separation of the assay mixture, containing 50 mM Tris-HCl, pH 7.5, 0.2 mM CMP, 1 mM MgCl2, 1 mM DTT, and 20 fiL of lysate in 0.5 mL of total volume (b) at time zero and (c) after 30 minutes of incubation (c). (From Amici et al., 1994.)...

Fig. 3-93. Separation of nucleosides using anion exchange chromatography. — Separator columns 2 IonPac AS4A eluent see Fig. 3-92 flow rate 1.5 mL/min detection suppressed conductivity injection volume 50 pL solute concentrations 10 ppm cytidine (1), 5 ppm adenosine (2), 10 ppm thymidine (3) and uridine (4), 13 ppm 2 -deoxyguanosine (5), 15 ppm guanosine (6), and inos-ine (7).

The apparent volume of distribution increases with increasing infusion time for infusions of under 70 minutes it is 501/m but increases to 3701/m for infusions lasting over 70 minutes the half-lives are 30-90 minutes and 4-10 hours respectively. Binding to plasma proteins is minimal. Liver elimination is driven by cytidine deaminase and results in the formation of an inactive metabolite, 2, 2 -difiuorodeoxycytidine (dFdU). About 95% of the dose is excreted in the urine (principally as dFdU) and 1% in the feces [39 ]. 

Figure 8 Enzymatic resolution of ( ) 10 (BCH189) at 90 g scale using free cytidine deaminase. Ninety grams of racemic 10 was incubated at 32 C with ISOO units of cytidine deaminase in a 15 liter volume in a 20 liter fermentor. The pH was adjusted to 7.0 with concentrated ammonia solu tion and maintained constant by the addition of 20% (v/v) acetic acid. At intervals, samples were analyzed by chiral HPLC [column. Cyclobond I acetyl (250 xO.46 mm) mobile phase, rriethylam-monium acetate (0.2% v/v), pH 5.5 flow rate, 1 inl/inin detection wavelength, 270 run]. (B) Epivit, g/liter (9) ( t-) 10, g/liter ( ) ee (Epivii) (T) conversion (% deamination of racemic 10). (Reproduced from Ref. 57 with the permisionofBurterwcMTh-Heineniann.)...

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